Matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI) is a relatively new and powerful technique able to study intact tissue sections providing ion distribution maps of many nonlabeled endogenous and exogenous species simultaneously. The absence of the label makes this technique fast, relatively inexpensive. The adoption of MALDI-MSI in skin research could contribute to the study of drug/toxicant absorption, drug/toxicant response, infections, wound healing and burn. In our study, a novel methodology for quantitative assessment of percutaneous absorption of an antifungal agent (Terbinafine hydrochloride) in a 3D skin model, Labskin, by MALDI-MSI has been presented. A quantitative assessment of the effect of adding a penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed. Quantitative MSI data demonstrated an increase of concentration of Terbinafine into the upper epidermis of Labskin in response to an increase of percentage of DMI in the delivery vehicle. In an additional study, MALDI-MSI was used to assess the metabolic activity in Labskin by employing the approach of "substrate-based mass spectrometry imaging" (SBMSI). Preliminary MALDI-MSI data detected the activity of the carboxylesterase 1 enzyme in the epidermal layer of skin. Thus, this research line aims to develop robust methods for a label-free determination of either drug absorption and drug biotransformation in skin, which are essential parts of pharmaceutical discovery and development.